Antigenic epitopes of IgE present on B cell but not basophil surface

ABSTRACT

Antigenic epitopes associated with the extracellular segment of the domain which anchors immunoglobulins to the B cell membrane are disclosed. For IgE, the epitopes are present on IgE-bearing B cells but not basophils or the secreted, soluble form of IgE. The epitope can be exploited for diagnosis.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 229,178, now abandoned, filed Aug. 5, 1988, which is acontinuation-in-part of U.S. patent application Ser. No. 226,421, July29, 1988, which is a continuation-in-part of U.S. patent applicationSer. No. 140,036 (now abandoned) filed Dec. 31, 1987.

BACKGROUND

The immediate-type hypersensitivity, such as extrinsic asthma, hayfever, and allergic responses to certain food or drugs, is mediatedprimarily by immunoglobulin E (IgE). In an IgE-mediated allergicresponse, the allergen binds to IgE on the surface of mast cells andbasophilic leukocytes (basophils). This binding causes a crosslinking ofthe IgE molecules and hence the underlying receptors for the Fc portionof IgE (FcεR) and thereby triggers the release of pharmacologicmediators, such as histamine, slow-reacting substance of anaphylaxis andserotonin. The release of these mast cell and basophil products causesthe various pathological manifestations of allergy.

IgE is produced by surface IgE-bearing B lymphocytes (B cells). Inindividuals sensitized to specific allergens, the allergen-specific IgEis produced by B cells continuously. Recently, Whitaker (U.S. Pat. No.4.714.759) described a method of treating allergy patients withtoxin-conjugated antibodies that were specific for IgE isotype. Theintended pharmacological effects of the immunotoxin is to killIgE-producing B cells.

In the U.S. patent application Ser. No. 140,036 (now abandoned), filedDec. 31, 1987 and in its Continuation-in-Part application Ser. No.07/226,421, filed Jul. 29, 1988, it was noted that IgE bound to thereceptors for the Fc of IgE (FcεR) on the surface of basophils and mastcells very strongly. The association constant, Ka, is in theneighborhood of 1×10¹⁰ liter/mole and the "off" time is more than 20hour. Even though IgE is not synthesized by basophils and mast cells,the very strong and stable association of IgE with FcεR makes IgEvirtually a surface antigen of these cells. It was indicated in ourapplications that an immunotherapeutical agent targeting the IgE on Bcells must not react with the IgE on basophils and mast cells.Antibodies which react with IgE isotype will cross-link IgE and theunderlying FcεR on basophils and mast cells and, when administered invivo, will induce systematic histamine release leading to anaphylaxis.

In the related patent applications mentioned above the development ofmonoclonal antibodies that recognized an antigenic epitope present onthe IgE on B cells but not on the IgE on basophils was described. Inaddition, the method of using the antibodies for treating allergy,either in the form of plain antibodies or toxin-conjugated antibodieswas described. The plain antibodies can cause the pharmacologicalmechanism of antibody-dependent cellular cytotoxicity (ADCC) and thetoxin-conjugated antibodies can directly cause cytolysis. Both of thesemechanisms can lead to the depletion of IgE-bearing B cells withoutharming the basophils and mast cells. The IgE epitopes present on Bcells but absent on basophils were termed ige.bl epitopes (bl stands forB lymphocytes).

SUMMARY OF THE INVENTION

The invention pertains to a new class of antigenic ige.bl epitopes (i.e.antigenic epitopes on B cells but not basophils) and the related classof antigenic epitopes which are present generally on B cellmembrane-bound immunoglobulins but not on the secreted, soluble form ofthe immunoglobulin. The invention also pertains to methods of exploitingthese newly discovered epitopes for therapeutic and diagnostic purposes.

B cells express on their surface antibody molecules which serve asreceptors for antigens during immunological induction. Themembrane-bound immunoglobulins differ from the secretory, solubleimmunoglobulins synthesized by the same cells in that they have an extrapeptidic piece that anchors the them onto the B cell surface. All theten membrane-bound immunoglobulins on B cells from different species,for which amino acid sequences have been determined, have extra isotypespecific regions that anchor the immunoglobulins to the membrane. Thesepeptidic regions have lengths ranging from 41 to 72 amino acids and canbe divided into three segments in terms of locations in relation to theplasma membrane. The middle segments of 25 hydrophobic and unchargedamino acid residues are in the membrane lipid bilayer; the C-terminalhydrophilic segments of 3-28 amino acid residues are intracellular; thesegments toward N-terminus contain 13 to 27 amino acid residues, arehighly acidic and hydrophilic and are on the extracellular surface ofthe plasma membrane. This part of membrane-bound region of mouse and ratIgE has 19 amino acid residues, among them, 10 are Glu or Asp residues.The length and the hydrophilic and highly charged nature of theextracellular segment indicate that this segment is exposed andaccessible to antibodies. These antigenic epitopes located on theextracellular segment of membrane-bound region of immunoglobulin heavychains can be generally designated mb/ec. The mb/ec segment of IgE canbe disignated ε.mb/ec segment.

The present invention pertains to the discovery of these mb/ec epitopes,particularly the ε.mb/ec epitopes, and their utility for immunotherapyfor of allergy. It also pertains to the development of monoclonal andpolyclonal antibodies against these epitopes, the use of theseantibodies, their immunoconjugates, and their parotope specificanti-idiotypic antibodies in treating allergy.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 outlines the process of determining mb/ec segment of human IgE(ε.mb/ec).

FIG. 2 shows DNA probes to be used for screening cDNA library for clonescontaining ε.mb/ec.

FIG. 3 shows the C-terminus of CH₄ domain of human E chain and thelocations of probe b and d.

DETAILED DESCRIPTION OF THE INVENTION 1. Unique Antigenic Epitope of IgEas targets of Immunotherapeutical Agents

IgE is present on three cell types in this body, IgE-producing B cells,mast cells, and basophils. If an antigenic epitope of IgE is present onB cells and not on basophils and mast cells, these epitopes (defined asige.bl) are virtually unique cell surface markers of IgE-bearing Bcells. These markers provide for several types of monoclonal orpolyclonal antibody-based therapy for IgE-mediated allergic diseases.

2. Anchoring Peptidic piece of B Cell Membrane-bound Immunoglobulins

B cells express on their surface antibody molecules, which serve asreceptors for antigens during immunological induction. Themembrane-bound immunoglobulins differ from the secretory immunoglobulinssynthesized by the same cells in that they have extra peptidic piecesthat anchor the immunoglobulin molecules onto the cell surface.

The amino acid sequence data of the ten membrane-bound immunoglobulinsfromseveral species have been determined. See Ishida, N. et al., EMBOJ., 1:1117 (1982); Steen, M. L. et al., J. Mol. Biol., 177:19-32 (1984);Rogers, J. et al., Cell, 26:19-27 (1981); Yamawaki-Kataoka, Y. et al.,Proc. Natl. Acad. Sci., MSA, 79:2008-2012 (1982); Kamaromy, M. et al.,Nuc. Acids Res., 11:6775-6785 (1983); Rogers, J. et al., Cell,20:303-312 (1980); Bernstein, K. E., J. Emmunol, 132:490-495 (1984);Cheng, H. et al., Nature, 296:410-415(1982). These sequences indicatecertain common features of the plasma membrane bound peptidic piece. Asshown in Table 1.the peptidic anchor piece has three segments which aredistinguishable based upon their locations in relation to the plasmamembrane. Even thoughthese peptidic pieces are short, ranging from 41 to72 amino acid residues,and have often been referred to as the"membrane-bound domain", the peptides are not entirely in the membranelipid bilayer. In fact, only 25 amino acid residues, largely hydrophobicresidues and threonine and serineresidues, located in the middle part ofthe peptides, are in the lipid bilayer. The C-terminal, hydrophilicsegments of 3 to 28 amino acid residues are located on the cytoplasmicside of the membrane. The segmentstoward the N-terminus, which areconnected to the third or fourth constant domains of the immunoglobulinheavy chains (CH₃ or CH₄) are veryhydrophilic and are on theextracellular side of the plasma membrane.

                                      TABLE 1                                     __________________________________________________________________________    Key features and properties of peptidic segments unique to                    membrane-bound immunoglobulins on B cells.                                    __________________________________________________________________________             First  Middle  Last                                                           segment                                                                              segment segment Total                                         __________________________________________________________________________    Immunoglobulin                                                                         Length                                                               Class/Subclass                                                                         # Amino acid residues                                                Mouse IgE                                                                              19     25      28      72                                            Rat IgE  19     25      28      72                                            Mouse IgG.sub.1                                                                        18     25      28      71                                            Mouse IgG.sub.2a                                                                       18     25      28      71                                            Mouse IgG.sub.2b                                                                       18     25      28      71                                            Mouse IgG.sub.3                                                                        18     25      28      71                                            Mouse IgM                                                                              13     25       3      41                                            Rabbit IgM                                                                             13     25       3      41                                            Human IgD                                                                              27     25       3      55                                            Mouse IgD                                                                              26     25       3      54                                            Properties                                                                             Hydrophilic                                                                          Hydrophobic                                                                           Hydrophilic                                                    Highly acidic                                                                        No charged                                                                    residues                                                      Physical On exterior                                                                          In membrane                                                                           On cytoplasmic                                        Location surface                                                                              lipid bilayer                                                                         surface                                               Abbreviated                                                                            mb/ec  mb/tm   mb/ic                                                 Symbols  segment                                                                              segment segment                                               __________________________________________________________________________    *mb for membranebound; ec for extracellular; tm for transmembrane; and ic      for intracellular.                                                       

The shortest length of the extracellular segment of the membrane-boundpieces of the immunoglobulins (designated mb/ec segments) has 13 aminoacid residues (Mouse and rabbit μ chains). See Table 2. The mb/ecsegments of all immunoglobulins contain high proportions of chargedamino acid residues, almost entirely acidic residues. The proportions ofchargedamino acid residues and polar hydrophilic residues account forvery high percentages of the amino acid composition of the mb/ec segment(Table 3). These parameters indicate that all the mb/ec segments areexposed and longenough to be accessible by antibodies. Studies of theevolution of immunoglobulin heavy chains indicate the ε and γ chains aremore related to each other (had more recent common ancestry) than tootherchains (Lin. L. C. and Putnam, F. W., Proc. Natl. Acad. Sci. USA,1981). Inaddition, the heavy chains evolved before the various mammalsspecies, mice, rats, and humans evolved. Thus, among the ten variousmb/ec segmentsthat have been determined, it is probably the murine orthe rat ε.mb/ec that the human ε.mb/ec, which sequence has not yetbeenreported, will be most related. The next most related will be the γchains. The murine or rat ε.mb/ec has 19 amino acid residues, among them8 Glu and 2 Asp residues. These data also provide support that humanε.mb/ec segment is exposed and accessible to antibodies.

                  TABLE 2                                                         ______________________________________                                        The amino acid sequences of the exterior portion of                           peptidic segments unique to membrane-bound                                    immunoglobulins (mb/ec segments).                                             Mb/ec segment                                                                        2621161161                                                             ______________________________________                                        Mouse IgE                                                                              ..ELDI.QDLCI.EEVEG.EELEE                                             Rat IgE  ELDI.QDLCT.EEVEG.EELEE                                               Mouse IgG.sub.1                                                                         GLQ.LDETC.AEAQD.GELDG                                               Mouse IgG.sub.2a                                                                        GLD.LDDVC.AEAQD.GELDG                                               Mouse IgG.sub.2b                                                                        GLD.LDDIC.AEAKD.GELDG                                               Mouse IgG.sub.3                                                                         ELE.LNGTC.AEAQD.GELDG                                               Mouse IgM                                                                              EGE.VNAEE.EGFEN                                                      Rabbit IgM                                                                             EGE.VNAEE.EGFEN                                                      Human IgD                                                                              YL.AMTPL.IPQSK.DENSD.DYTTF.DDVGS                                     Mouse IgD                                                                              I.VNTIQ.HSCIM.DEQSD.SYMDL.EEENG                                      ______________________________________                                    

                                      TABLE 3                                     __________________________________________________________________________    Composition of charged amino acid residues and polar,                         hydrophilic amino acid residues in the exterior portion of                    peptidic segments unique to membrane-bound immunoglobulins                    (mb/ec segments).                                                                                              Proportion                                                              Total of hydro-                                                Acidic                                                                             Basic                                                                              Polar                                                                              hydrophilic                                                                         philic                                              TOTAL                                                                              residues                                                                           residues                                                                           residues                                                                           residues                                                                            residues                                     __________________________________________________________________________           # Amino acid residues     %                                            Mouse IgE                                                                            19   10   0    2    12    63                                           Rat IgE                                                                              19   10   0    2    12    63                                           Mouse IgG.sub.1                                                                      18   6    0    4    10    56                                           Mouse IgG.sub.2a                                                                     18   7    0    2    9     50                                           Mouse IgG.sub.2b                                                                     18   7    1    1    9     50                                           Mouse IgG.sub.3                                                                      18   6    0    4    10    56                                           Mouse IgM                                                                            13   6    0    2    8     61                                           Rabbit IgM                                                                           13   6    0    1    7     54                                           Human IgD                                                                            27   6    1    8    15    56                                           Mouse IgD                                                                            26   7    0.5  9    16.5  63                                           __________________________________________________________________________    Acidic residues: E (Glu), D (Asp)                                             Basic residues: K (Lys), R (Arg), H (His); His is partially charged.          Polar residues: S (Ser), T (Thr), C (Cys), Q (Glyn), N (Asn)              

3. Determining the amino acid sequence of mb/ec segment of human Ig(Eε.mb/ec segment)

A number of well established procedures can be applied to determine theDNAsequence corresponding to the human ε.mb/ec segment. In one approach(FIG. 1), the starting point is the mRNA preparation of a human myelomacell line which express IgE on the surface. SK007 cells can be employedfor this purpose. With the mRNA preparation, one can establish a cDNAlibrary employing cloning vector with λ phage or plasmids. A preferredmethod for constructing the cDNA library is with the cDNA LibraryConstruction System Kit--Librarian I developed and commercialized byInvitrogen (San Diego, Calif.). Stepwise detailed instruction manualisprovided for RNA isolation from cells, reverse transcription, secondstrandsynthesis, linker ligation, agarose gel sizing of cDNA,electroelution to purify cDNA, vector ligation, and transformation of E.coli. The vector used in this library is pCDM8.

In the screening of the cDNA library for clones containing the ε.mb/ecsegment, several probes can be used. As shown in FIG. 2, the library canbe screened with DNA probe a, which is a 1.1 kb long U266 cDNA coveringmost of length of ε mRNA (no membrane-bound segment). The positiveclones which include both secreted and membrane-bound forms can bedistinguished by using additional probes. Probe b is developed by takingadvantage of the very likely property that the end of the CH₄ domain istruncated in the human ε chain of the membrane-bound form. Thetruncation occurs when the gene segments of CH₄ domain and membranebound domain are translocated. The loss ofC-termini occur with themembrane bound forms of immunoglobulins, includingand μ, which containCH₄ domains From the published information on nucleotide sequence ofhuman ε.CH₄ domain, the most possible splicing donor site is intracodonGT, 71 bp 5' of the termination codon TGA. Another GT, which is notintracodon and less likely a splicing donor site, is closer to theterminus (24 bp 5' to the termination codon).

The specific location for probe b is indicated in FIG. 2 and FIG. 3. Theprobe will react with secreted form of ε chain gene and not membraneform of ε chain gene.

The design of probe c is based on the finding that the transmembranesegment of the membrane-bound domain (mb/tm segment) is very conservedamong all the immunoglobulin genes so far sequenced. There is a segmentofpeptide and its corresponding coding DNA within this mb/tm segment,that isnearly identical among all immunoglobulins. As shown in Table 4,the consensus DNA sequence with the eight combinations will be used asprobe c. FIG. 2 shows the location of the probe.

                  TABLE 4                                                         ______________________________________                                        A conserved region in the transmembrane portion of the                        peptidic segment of the membrane-bound                                        immunoglobulins (in mb/tm segment.                                                          12345                                                                         Leu.Phe.Leu.Leu.Ser.                                            ______________________________________                                        Mouse IgE       CTG.TTC.CTG.CTC.AG                                            Rat IgE         CTG.TTC.CRG.CTC.AG                                            Mouse IgG.sub.1 CTC.TTC.CTG.CTC.AG                                            Mouse IgG.sub.2a                                                                              CTC.TTC.CTG.CTC.AG                                            Mouse IgG.sub.2b                                                                              CTC.TTC.CTG.CTC.AG                                            Mouse IgG.sub.3 CTC.TTC.CTG.CTC.AG                                            Mouse IgM       CTC.TTC.CTC.CTG.AG                                            Rabbit IgM      CTG.TTC.CTG.CTG.AG                                            Human IgD*      CTC.TTC.ATC.CTC.AC                                            Mouse IgD*      CTC.TTC.CTG.CTC.AC                                            Consensus sequence (Probe c)                                                                   ##STR1##                                                     ______________________________________                                        *Human and mouse IgD's have Thr (ACXO in the 5th amino acid residue; human     IgD also has Ile (ATC) in the 3rd amino acid residue. These are the           variations not covered by the consensus sequence.                        

Probe d which represents a segment upstream to the most possiblesplicing donor site, GT, is consisted of 36 bp (FIG. 2 and FIG. 3). Thisprobe should react with ε chain gene of both secreted andmembrane-boundforms.

Table 5 summarizes the pattern of reactivities of clones containing εgenes of secreted or membrane-bound forms with the four probes.

                  TABLE 5                                                         ______________________________________                                        The reactivity of ε gene-containing cDNA                              clones with probes a, b, c, and d.                                                      ε Secreted                                                                    ε Membrane-bound                                    ______________________________________                                        Probe a     +         +                                                       Probe b     +         -                                                       Probe c     -         +                                                       Probe d     +         +                                                       ______________________________________                                    

The library size needed to clone the membrane bound ε chain dependsonhow abundant the mRNA is. Assuming secreted IgE comprises 0.1% of theSK007 poly A⁺ RNA, the library size should be about 5.000independentrecombinant clones to have a 99% possibility to isolate apositive clone. In IgE producing rat immunocytoma IR2 and IR162 cells,mRNA for the membrane-bound form of ε chain was found to be more than 2%of that of the secreted form. Assuming this ratio ofmembrane-bound/secreted forms of ε chain holds true for the humanIgE-producing SK007 cells, the cDNA library size needed to isolate themembrane-bound ε chain is about 250.000. In a preferred procedure, alarger number of clones, 1,000,000, are screened.

An alternative to the conventional approach of establishing cDNA libraryand screening the clones representing the cellular mRNA species is toamplify the mRNA to produce high proportions of their corresponding DNA.The resulting DNA can then be purified by gel electrophoresis and thensubjected to sequence analysis. The methodology, referred to aspolymerasechain reaction (PCR) amplification, has been established inthe past few years and complete system including reagents and equipmentshave been commercialized. One preferred system is provided by PerkinElmer Cetus (Norwalk, Conn.). The reagents kit is the GeneAmp DNAAmplification Reagent Kit and the equipment is the DNA Thermal Cycler.

Some of the specific reagents used in this approach are the same as usedfor the cDNA library cloning. Since no sequence in the membrane boundsegment of the ε chain has been determined, the strategy is to amplifyboth the secreted and membrane-bound forms of ε chains. Two primers areto be used, one is oligo.dT (25-30-mers) and one is the oligomercorresponding to probe d in FIG. 2 and FIG. 3. Probe d is located5' tothe most possible splicing donor site and therefore primes both thesecreted and membrane-bound forms of ε mRNA and DNA. After sufficientamplification, the two populations of DNA fragments are resolved by gelelectrophoresis. The secreted form of the e chain can be distinguishedby its reactivity with probe b The purified DNA's are then subjected toDNA sequencing.

PCR amplification seems to be more efficient procedure than cDNA cloningfor mRNA poorly represented in the poly A⁺ RNA pool. The U266 ε chaincDNA can be used to work out some preliminary annealing conditionsbetween template DNA and oligo-primers.

Another approach of obtaining a DNA clone containing genes encoding themembrane bound segments is to screen human genomic DNA library. Humangenomic DNA library is readily available. A preferred source is thelibrary constructed using human lung fibroblast WI38 cells provided byStratogene (La Jolla, Calif.). The genes are in λ vector and theinserted DNA have average sizes of 15K bp. Identification of the clonescan be achieved by hybridization with U266 cDNA clone DNA. The locationofthe gene segment corresponding to the membrane bound region can bedetermined by using probe prepared from the homologous mouse gene of thetransmembrane segment (probe c of FIG. 2 and Table 4). The sequence ofthemembrane-bound segment is then determined.

3A. The nucleotide sequence of DNA encoding membrane anchoring peptideof human ε chain

Nucleotide sequence of genomic DNA encompassing the encoding segmentsfor the membrane anchoring peptide of human membrane bound s chain wasdetermined and is shown below along with the deduced amino acid sequencefor portions of the membrane anchoring peptide. The assignment of theexons was made by identifying the nucleotides for splicing donors andacceptors and by comparing to the published homologous sequences ofmouse membrane bound ε chain and of immunoglobulins of other classes.##STR2##

Human ε.mb/ec peptide is identified as the first fourteen amino acidsencoded by membrane exon I. This precedes a stretch of about 25hydrophobic amino acids which are the transmembrane region. Twopossibles structures of ε.mb/ec are shown below.

Possible Structures of Human ε.mb/ec peptide ##STR3##As described morefully below, the ε.mb/ec peptide can be used to elicit antibodies whichreact specifically with membrane bound immunoglobulin E. For thispurpose, the peptides can be chemically synthesized by standardtechniques of protein synthesis. A preferred method for synthesizing thepeptides is with RaMP system (DuPont, Wilmington, Del.), which appliesFmoc chemistry. Alternatively, the proteins can be biosynthesized byemploying oligodeoxynucleotides encodingthe peptide. The nucleotidesequence is given as the 5' portion of membraneexon I, above.

As immunogens, the proteins may be used in either the monomeric ordimeric structural forms shown above. Peptides comprising the humanε.mb/ec segment and the connecting four amino acids in the CH₄ domaincan also be used. In addition, modified peptides having substantialimmunological equivalency can be used. For example, the peptide aminoacid sequence shown above can be modified by deletion, insertion orsubstitution of one or more amino acids which do not essentially detractfrom the immunological properties of the peptide. The peptides can alsobe used as polymers where the amino acid sequence shown above, orequivalent sequence, is the polymer repeat unit.

4. Developing Antibodies to mb/ec Segment

The ε.mb/ec peptide can be used in the immunization of animals toprepare polyclonal and monoclonal antibodies. They can also be used toscreen for specific monoclonal antibodies or characterize specificpolyclonal antibodies. They can also be used to purify monoclonal andpolyclonal antibodies.

In the process of preparing for monoclonal antibodies specific forε.mb/ec peptide, it is not necessary to use the ε.mb/ec peptide in bothimmunization and antibody identification. For example, in immunizingmice for preparing immune spleen cells for fusion with myeloma cells,the immunogen may be the membrane-bound IgE isolated from plasmamembrane of IgE-bearing myeloma cells, such as SK007 cells. Theimmunogen may also be the IgE-bearing myeloma cells.

For using the synthetic ε.mb/ec peptide for immunogen, it is moreeffective to conjugate the peptide to a protein carrier. A preferredprotein carrier is keyhole lympit hemocyanin (KLH). If the peptidicsegment lacks a Lys residue or if the Lys residue is in the middle partofthe segment, it is desirable to add a Lys residue at the C-terminalend. Because the N-terminus already has an α-amino group, the modifiedsynthetic peptidic will have two amino groups for linking.

Multiple molecules of peptides can be con jugated to each molecule ofthe carrier protein. With KLH, a preferred molar ratio for peptide/KLHis 10. The method of conjugation is very well established. Cross linkerssuch as glutaldehyde or bis (sulfosuccinimidyl) suberate ordisulfosuccinimidyl tartarate (Catalogue #21579, 20591, Pierce ChemicalCo., Rockford. Ill.) have been used. A preferred cross-linker is thelatter.

The immunogen, such as the KLH conjugate, can be used to immunizerabbits, goats, rats, or mice to prepare polyclonal antibodies specificfor the ε.mb/ec peptide. Lympocytes from the spleen or lymph nodes ofimmune mice and rats can also be taken to prepare hybridomas secretingmonoclonal antibodies specific for the ε.mb/ec peptide. A preferredprotocol to prepare the monoclonal antibodies is to fuse immune spleencells of mice with non-secreting mouse myeloma cells, such as NS-1 orSP2/0 cells using polyethylene glycol.

For optimal immunization of mice, 50 μg of the peptide-KLH conjugate incomplete Fruend adjuvant is injected subcutaneously into each mouse forpriming. Two and four weeks later, same amounts of antigen is given s.c.in incomplete Fruend adjuvant. At about the six week time point, thefourth antigen injection is given i.p. in saline. Mice are sacrificed 4days after the last injection and the spleens are taken for preparingsingle cell suspension for fusion with myeloma cells. Similar protocolcanalso be used for immunization with purified native humanmembrane-bound IgE(having attached membrane anchor domain) isolated fromthe plasma membrane of IgE-bearing human myeloma cells, such as SK007cells. When human IgE-bearing cells are used as the immunogen, 1×10⁷cells are injected i.p. with two week intervals.

The fusion procedure with polyethylene glycol and other variousprocedures concerning cloning and hybridoma culturing have been wellestablished and the preferred protocol is the same as described byHudson. L. and Hay. F C. (Practical Immunology, 2nd edition, pp.303-313, 1980, Blackwell Publishing Co., Boston).

The screening of hybridomas for monoclonal antibodies or theidentificationof polyclonal antibodies reactive with ε.mb/ec peptide canbe performed with enzyme linked immunosorbent assays (ELISA) using thesynthetic ε.mb/ec peptide as the solid phase antigen. An alternativesolid phase antigen is the conjugate of ε.mb/ec peptide with a differentcarrier protein such as bovine serum albumin different from that used inimmunogen. Further characterization of the monoclonal and polyclonalantibodies are shown in Table 6. The assays employed in these studiesare also indicated. The assays have been described in detail in the U.S.patent application Ser. No. 226,421, filedJul. 29. 1988, and U.S. patentapplication Ser. No. 140,036 (now abandoned), filed Dec. 31, 1987. theteachings of which are incorporated by reference herein.

                  TABLE 6                                                         ______________________________________                                        The reactivity of antibodies specific for ε. mb/ec peptide            with different IgE-containing targets.                                                     Reac-                                                                         tivity                                                                              Assays                                                     ______________________________________                                        Synthetic ε. mb/ec peptide                                                           +       ELISA                                                  Soluble IgE    -       ELISA                                                  Basophils and mast cells                                                                     -       Immunofluorescence staining                                                   Histamine release                                      SK007 myeloma cells                                                                          +       Immunofluorescence staining                            IgE-bearing B cells                                                                          +       Immunofluorescence staining                            ______________________________________                                    

5. Experiments with Animal Models

The substances and methods are tested on animal model systems. Two ofthe most relevent systems are the following.

A. Asthma/rhesus monkey model

The monoclonal antibodies specific for human ε.mb/ec peptide and theirrelated substances of this invention are intended for use to treatpatients with various IgE-mediated allergies (see section 6 below).Among these allergies, extrinsic asthma is a more serious form. Anexperimental model system for studying asthma has been established withrhesus monkeys.

A small portion of rhesus monkeys, which have been infected with thenematode, Ascaris suum, develop sensitivity to extract of ascaris. Whenthese sensitive monkeys are given spray containing ascaris antigen, theydevelop breathing problems resembling asthma. Patterson, R., J. Clini.Invest. 57:586-593 (1976).

The various substance of this invention can be experimented in theasthma/rhesus monkey model system. The ascaris sensitive monkeys aregiventhe experimental treatment or control treatment and measurementsare made to determine:

(a) Does the asthma symptoms upon ascaris challenge decline?

(b) Does the circulating IgE decline?

(c) Does the circulating IgE-bearing B cells decline?

(d) Does the IgE density on basophils decline?

B. Mouse model system

Mice are not known to develop allergic symptoms naturally. However, fordemonstrating the pharmacologic mechanisms of the intended therapy inregards to the depletion of IgE-bearing B cells and IgE, the mouse canserve as an excellent model.

The ε.mb/ec segment of mouse has already been sequenced. Ishida, N.etal., EMBO J.. 1:1117-1123 (1982). The 19 amino acid residue peptide is

    Glu-Leu-Asp-Leu-Gln-Asp-Leu-Cys-Ile-Glu-Glu-Val -Glu-Gly-Glu-Glu-Leu-Glu-Glu

The peptide is synthesized in several forms, including one that hasextra Leu.Lys residues at the C-terminus.

The peptide and its KLH conjugate are used as antigens to immunizerabbits and goats. The antisera are collected. The antigen-specificantibodies arepurified using column of Sepharose 4B conjugated with thepeptide (with Leu-Lys addition) or with peptide linked to bovine serumalbumin. Normal mice are injected i.v. or i.p. with the purifiedantibodies or their related substances to study the following questions:

(a) Does the total IgE in circulation decline?

(b) Does the number of IgE-bearing B cells decline?

(c) Does the density of IgE on the surface of basophils decline?

(d) Do IgM and IgG specific for ε.mb/ec peptide cause different effects?The purpose of this question is to address the effects ofantibody-dependent cellular cytotoxicity (ADCC) in the depletion ofIgE-bearing B cells. IgG, not IgM is known to mediate ADCC.

6. Therapy of IgE-mediated Allergy based upon the Selective Eliminationof IgE-producing Cells

Antibodies specific for the ε.mb/ec epitopes bind IgE on the surface ofIgE-producing B cells and not on basophils and mast cells.Thisdifferential binding of IgE-bearing cell types provides the basisfor therapeutic uses of the antibodies.

One of the most powerful agents that trigger the release ofpharmacologicalmediators of allergy from mast cells and basophils isanti-IgE antibody. Conventional anti-IgE antibody will bind IgE on thesurface of mast cells and basophils and trigger the release ofpharmacological mediators of allergy. The antibodies of this inventioncannot bind IgE on these cells.

A. Antibodies specific for IgE-producing cells

The antibodies specific for ε.mb/ec epitopes in the form of a murineantibody or in the form of mouse/human chimeric antibody, may be appliedin several ways for the treatment of IgE-mediated allergies. Theantibody can be used as an effector agent mediating an immune functionor as a carrier agent of toxins or cytotoxic drugs, as set forth below,for delivering an effector substance.

Antibodies of certain IgG subclasses, such as mouse IgG2a and human IgG1and IgG3, can mediate antibody-dependent cellular cytotoxicity (ADCC)carried out by certain Fc receptor-bearing phagocytic leukocytes. Theantibodies of this invention, especially in the form of mouse gamma 2aantibodies or chimeric antibodies bearing human gamma-1 or gamma-3chains,can be used to deplete IgE-bearing B cells by the ADCC mechanism.The antibodies can be administered as free antibodies to patientsafflicted with IgE-mediated allergy in amounts sufficient to eliminatesubstantiallyIgE-producing cells and consequently, to eliminatesubstantially IgE.

For therapeutic uses described, chimeric or "near-human" antibodies arepreferred. Chimeric antibodies comprise a variable or antigen binding(hypervariable or complementarity determining) region derived from ananimal antibody and the remaining regions derived from a human antibody.Methods for producing chimeric (e.g. murine/human) antibodies are wellestablished. Chimeric antibodies can be produced in large quantities andthey are less immunogenic in humans than nonhuman antibodies.Consequently, they are better suited for in vivo administration,especially when repeated or long term administration is necessary.Antibody fragments of the chimeric antibodies can also be used.

Immunotherapies employing the antibodies of this invention may be usedin combination with conventional desensitization immunotherapy. Forexample, desensitization with allergen may be performed in conjunctionwith the administration of anti-ε.mb/ec antibodies or immunotoxins (seeC section below) to eliminate substantially IgE-producing cells. Onemajor effect of desensitization is that IgG's are induced against theallergen/immunogen. The induction of an IgG response may be mosteffectivewhen IgE-producing B cells are substantially depleted. Thecombination of antibody and desensitization therapy is an attractiveform of therapy. IgE-producing B cells may be temporarily depleted (fora few weeks or months) by the anti-ε.mb/ec antibody and will eventuallyrepopulate. The desensitization may have longer lasting effects.

B. Immunotherapy combining an ε.mb/ec-specific antibody and a factorenhancing ADCC

Many factors, such as GM-CSF (granulocyte monocyte-colony stimulationfactor) or M-CSF (monocyte-colony stimulation factor), are known toinducethe proliferation of leukocytes, including those mediating ADCC.In in vitro experiments, GM-CSF and M-CSF have been shown to augment theADCC activity on tumor cells mediated by monoclonal antibodies specificfor surface antigens expressed on the tumor cells. It is conceivablethat the therapeutical effect of ε.mb/ec specific monoclonal antibodiesin treating allergies can be enhanced by combining the use of factorsthat augment ADCC activities.

C. Immunotoxins specific for IgE-producing cells

Antibodies specific for an ε.mb/ec epitope can be used as immunotoxinsspecifically targeted to IgE-producing B cells. The immunotoxin binds toIgE-producing B cells but not to mast cells or basophils. In this way,IgE-producing B cells can be selectively eliminated in a patientsuffering from an IgE-mediated allergy. The elimination of the Igproducing cells reduces IgE levels in the circulation which results in areduction of the amount of IgE available tobind mast cells andbasophils. The immunotoxin does not kill mast cells or basophils andcause the release of pharmacologic mediators from these cells.

Immunotoxins for selective binding to IgE-producing lymphocytes arecomprised of cytolytic or cytotoxic agents conjugated monoclonalanti-ε.mb/ec antibodies. The cytolytic agents can be selected fromany ofthe available substances including ricin, Pseudomonas toxin, diptheriatoxin, pokeweed antiviral peptide, tricathecums, radioactive nuclidesand membrane-lytic enzymes. The antibody and the cytotoxin canbeconjugated by chemical or by genetic engineering techniques.

The immunotoxins are administered to a patient afflicted withIgE-mediated allergy in amounts sufficient to reduce or to eliminateIgE-producing lymphocytes in the patient and thereby prevent oralleviate the symptoms of the IgE-mediated allergy. The immunotoxins maybe used alone or in combination with free anti-IgE antibody.

7. Antiidiotypic Antibodies and Methods of Active Immunization Againstε.mb/ec Epitope

The ε.mb/ec specific monoclonal antibodies described thus far can beused to generate parotope specific anti idiotypic antibodies which offeranother mode of treating IgE-mediated allergy. Antibodies against theparotope of the ε.mb/ec specific antibodies conformationally resemblethe epitope for which the anti-IgE antibody is specific, that is,theyresemble an ε.mb/ec epitope. These anti-idiotypic antibodies can be usedto actively immunize against ε.mb/ec and induce the endogenous formationof antibodies against the ε.mb/ec epitope. The induced antibodies willmediate the various therapeutical effects of ε.mb/ec-specificantibodies.

Because an ε.mb/ec epitope is a "self-molecule", it is not immunogenic.However, active immunization against it may be achieved by using theparotope-specific antibodies of this invention. The parotype-specificantibody shares conformational resemblance with the antigen--the ε.mb/ecepitope--which can elicit immune response in humans against the epitope.

Paratope-specific, anti-idiotyptic antibodies are administered to apatientsuffering from IgE-mediated allergy in an immunogenic amount toinduce the formation of ε.mb/ec antibodies. The anti-idiotypicantibodies arepreferably administered as chimeric antibodies. They mayalso be given as antibody fragments (which also may be chimeric innature).

8. ε.mb/ec Peptide Analogues and Active Immunization Against ε.mb/ecEpitope

Even though human ε.mb/ec peptide is probably not immunogenic in humans,peptide with the same sequence and amino acid substitutions canbeimmunogenic and induce antibodies that cross react with authenticε.mb/ec epitope. These ε.mb/ec peptide analogues can be administered topatients suffering IgE-mediated allergies. The antibodies induced bythis active immunization can achieve the functions as the antibodiesdescribed in section 6.

9. Diagnostic Uses

Antibodies against ε.mb/ec epitopes can be used to identify andenumerate IgE-bearing lymphocytes in mixed leukocyte populations. Forthispurpose, antibodies can be used in standard assay formats fordetermining cell surface antigens. In general, the antibody is contactedwith a sampleof the leukocytes to be tested under conditions which allowthe antibody tobind IgE-bearing cells in the sample. The cells are thenexamined for binding of antibody. This can be accomplished byconventional cell staining procedures. For example, a fluoroscentlylabeled second antibody can be used to detect binding of the anti-IgEantibody.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

I claim:
 1. An antibody which specifically binds to the extracellular segment of the membrane-bound domain of a membrane-bound immunoglobulin chain.
 2. A monoclonal antibody which specifically binds to the extracellular segment of the membrane-bound domain of a human membrane-bound immunoglobulin chain.
 3. An antibody of claim 1, wherein the immunoglobulin is human IgE.
 4. An antibody of claim 1, which is monoclonal.
 5. Monoclonal antibody which specifically binds to the extracellular segment of the membrane-bound domain of human IgE but does not bind to human IgE on the surface of basophils and does not bind to secreted, soluble IgE.
 6. Monoclonal antibody of claim 5, which is a mouse IgG2A subclass antibody.
 7. A continuous stable cell line which produces a monoclonal antibody which specifically binds to the extracellular segment of the membrane-bound domain of human IgE but does not bind to human IgE on the surface of basophils and does not bind to secreted, soluble IgE.
 8. A cell line of claim 7, which is a hybridoma.
 9. A cell line of claim 8, which is a murine hybridoma.
 10. An antibody that specifically binds to a peptide having the amino acid sequence

    Glu Leu Asp Val Cys Val Glu Glu ala Glu Gly Glu Ala Pro

and modifications of the peptide in which amino acids have been deleted, inserted or substituted without detracting from the immunological properties thereof.
 11. An antibody of claim 10, which is monoclonal. 